Bacteria have employed several mechanisms to survive in the diverse environments they encounter. One such mechanism is the ability to form a biofilm, a bacterial community consisting of both viable and dead bacteria enmeshed in a complex of protein, nucleic acids, lipids and carbohydrates. Biofilm formation begins with the attachment of bacteria to a surface, the production of extracellular polymeric substances by the bacteria, and maturation of biofilm as other microbes and materials are added to the matrix. Microorganisms are known to be capable of adhering to any surface, including stainless steel, teflon, the widely used plastic polyvinyl chloride (PVC), and even the high grade plastic material polyvinylidene fluoride (PVDF). As such, bacteria can form biofilms on any number of substrates in industrial, marine, military, and residential settings including water and sewage pipes, food processing equipment, cooling or heating water systems, fuel systems, off shore oil and gas pipelines, boat hulls, toilets, drains, and sinks. In many cases biofilms formed on these surfaces causes corrosion of materials which require costly repair and are a hazard to environmental and personal safety. Further, biofilms can pose an infectious threat when bacteria disperse from the biofilm into food and water supplies. Biofilm-forming bacteria are also a serious threat in medical environments where they are commonly found on tubing, catheters, stents, prostheses and heart valves. Moreover, bacterial biofilms can also found on bone, teeth, and other tissues and by one estimate, are responsible for 80% of all infections of the human body that include middle ear infections, gingivitis, endocarditis, and urinary tract infections.
The ability of bacteria to form a biofilm enables microorganisms to adhere, persist, and even thrive on countless materials in diverse environments. The biofilm matrix protects embedded bacteria from changes in pH, temperature, nutrient and oxygen content. The biofilm matrix also induces dramatic changes in bacterial gene regulation, metabolism, and resistance that facilitate bacterial growth and survival in adverse conditions. Biofilm bacteria are also extremely resistant to biocides used in medical, industrial, residential and military settings to decontaminate equipment, infrastructure, and devices as well as to treat infected patients. Investigators have reported that biofilm bacteria were 150 to more than 3000 times more resistant to hypochlorous acid (free chlorine, pH 7.0) than planktonic bacteria. The food industry reported similar findings, which necessitate the use of high concentrations of chlorine to rid processing facilities of contaminating biofilm-forming microbes and the need to find better disinfectants. Not surprisingly, the biofilm matrix also impedes host-mediated response to infection, and confers resistance to an array of antibiotics typically used to treat bacterial infections.
Three of the most problematic opportunistic bacteria associated with highly refractory infections in hospitals and clinics around the world are Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus epidermidis. P. aeruginosa, a Gram-negative bacterium, ranks fourth as the most common nosocomial pathogen accounting for approximately ten percent of all hospital-acquired infections (41). A. baumannii, a Gram-negative bacterium, found in intensive care type settings where immune-compromised patients are intubated, have multiple intravenous lines or monitoring devices, surgical drains, or indwelling urinary catheters. A. baumannii has been particularly problematic in infections of wounded U.S. military personnel over the past decade during the Iraq and Afghanistan war, and has also been reported in water pipes. S. epidermidis is a common Gram-positive organism that comprises the majority of the human commensal skin microflora, and depending on the strain and the immune status of the patient, can also cause severe hospital-acquired infections, primarily associated with indwelling medical devices. Staphylococcal species have been identified as contaminates in drinking water, on food processing equipment and other environmental surfaces.
Biofilms of the “Mode I” variety are commonly known as surfaced-attached microbial communities that are sculpted strategically by microbes embedded within them as highly complex structures composed of well-developed matrices of live and dead organisms, as well as polysaccharides, nucleic acids, lipids and proteins. Mode II biofilms are not surface-attached and comprised of organisms such as P. aeruginosa that are attached to one another in macrocolonies enmeshed in thick, highly inspissated mucus in airway diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) Pseudomonas biofilms can also be found growing on medical devices, in water and sewage pipes, in swimming pools fuels, and fuel systems.
As the cells within a biofilm differentiate and a biofilm matures, reduced metabolic rates, the cellular expression of defense mechanisms and the reduced ability of antimicrobial agents to penetrate the biofilm result in increased antimicrobial resistance and make a biofilm particularly difficult to eradicate. Present biofilm control strategies typically target the early stages of biofilm development and involve the use of toxic antimicrobial agents. However such toxic agents present their own downstream problems due to their release into the environment.
A chemical library is a collection of stored chemicals typically generated, for example, from specific synthetic or functional goals, or from a series of untargeted synthetic efforts, and/or from purchases and acquisitions of smaller libraries. Each chemical in a library has associated information stored in some kind of database, such as the chemical structure, purity, quantity, and physiochemical characteristics of the compound. Many thousands of well-categorized chemical libraries are known to exist and such libraries have found particular use in high through-put screening in the drug development industry.
The development of high-through put screening platforms for identifying agents effective against biofilms, however, poses particular challenges. The matrix-encased biofilm is specialized for surface persistence and biofilms may be highly resistant to antibiotics effective against planktonic bacteria of the same species. The matrix itself may provide a barrier to penetration by a biocide. The individual bacterial cells are typically metabolically less active than their dispersed counterparts, and therefore may be less susceptible to effects of an anti-microbial agent. Cells in a biofilm are also thought to develop a protected biofilm phenotype, for example by up-regulation and increased expression of drug efflux pumps. Hence, modalities which are effective against planktonic bacteria, and which are most amenable to testing and filtering via high through-put assay platforms, may have substantially reduced efficacy or be completely ineffective against the same bacteria residing in a biofilm.
Inhibition of biofilm formation may be effectively achieved by interfering with or preventing adhesion; however an inhibiting agent may be completely ineffective against an established biofilm and may in fact exhibit very little antibiotic activity. An antibiotic agent may be very effective at inhibiting biofilm formation simply by controlling the bacterial population, but may have little or no impact on an established biofilm. Further, an agent may exhibit efficacy with respect to killing an established biofilms by mechanisms relating to interference with colonization or association of bacterial cells, such as by triggering dispersal rendering dispersed bacteria vulnerable to antibiotics effective against planktonic cells, and yet the same agent may not exhibit any antibiotic activity itself.
Further, bacterial biofilms are often comprised of more than one species of bacteria. Agents capable of inhibiting biofilm formation of more than one species known to co-reside in biofilms are particularly desirable, and yet non-toxic agents capable of killing established biofilm colonies formed from multiple bacterial species are virtually unknown.
There remains a need for safe, non-toxic agents having efficacy in inhibiting biofilm formation and/or killing established bacterial biofilm. Clearly it would be advantageous to develop high-through methods for screening libraries of compounds to identify novel agents effective for inhibiting bacterial biofilm formation and/or killing established bacterial biofilm.